Method test media and chromogenic compounds for identifying and differentiating general coliforms and Escherichia coli bacteria

ABSTRACT

A chromogenic β-galactosidase substrate producing an insoluble precipitate of a first color when reacted upon by β-galactosidase and a chromogenic β-glucuronidase substrate producing an insoluble precipitate of a second, contrasting color when reacted upon by β-glucuronidase are combined in a test medium for quantitatively identifying and differentiating general coliforms and E. coli. The β-galactosidase substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside which produces an indigo blue precipitate when reacted upon by β-galactosidase may be used with one of the novel compounds 6-chloroindolyl-β-D-glucuronide, 4,6-dichloroindolyl-β-D-glucuronide, 6,7-dichloroindolyl-β-D-glucuronide, and 4,6,7-trichloroindolyl-β-D-glucuronide, which produce mauve or magenta precipitates when reacted upon by β-glucuronidase. The β-glucuronidase substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, which produces an indigo blue precipitate when reacted upon by β-glucuronidase may be used with one of the novel compounds 6-chloroindolyl-β-D-galactoside, 4,6-dichloroindolyl-β-D-galactoside, 6,7-dichloroindolyl-β-D-galactoside, and 4,6,7-trichloroindolyl-β-D-galactoside which produce mauve or magenta precipitates when reacted upon by β-galactosidase.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method, test medium, and novelchromogenic compounds for quantitatively identifying and differentiatinggeneral coliforms and Escherichia coli.

2. Description of the Related Art

Currently, in microbiology, the presence of indicator organisms iswidely used to determine the quality of various products. For example,in the analysis of water, food and dairy products, the presence ofmembers of the "coliform" group as well as the presence of the bacterialspecies Escherichia coli are considered very significant qualityindicators. Therefore, test methods to effectively identify andenumerate these bacterial types are needed, and there is a continuingsearch for better, more accurate and simpler test methods in this area.

Numerous methods for determining, identifying and enumerating coliformsand E. coli currently exist, with varying degrees of accuracy andfacility. Some test methods only indicate the presence or absence (P/A)of the organisms while some methods attempt to quantify the organisms inthe test materials. Following are some of the current methods.

Violet Red Bile Agar (VRBA)

This medium incorporates bile salts to inhibit non-coliforms. It alsocontains lactose with the pH indicator neutral red. As coliforms(especially E. coli) grow in the medium, the lactose is fermented withacid production and the neutral red in the area of the bacterial colonybecomes a brick red color. Therefore, any colonies growing as a redcolor in 24-48 hours are considered to be coliforms. This medium is noteasy to interpret and for E. coli quantification needs to be followed upby confirming tests such as brilliant green lactose broth fermentationor streaking on Eosin Methylene Blue Agar (EMBA). In spite of theseshortcomings, VRBA is an approved method for testing dairy products.

The Most Probable Number (MPN) method

This method utilizes various broth (liquid) media in tubes. Samples tobe tested are added in varying amounts to the broth media and afterincubation, the tubes are checked for growth and gas formation.Estimates of the numbers (populations) of bacteria are determined frompre-existing tables. The method is in general use, but the results aregiven in a general range and therefore are not very precise.

The Membrane Filter (MF) method

This method utilizes micropore filters through which samples are passedso that the bacteria are retained on the surface of the filter. It isused most often when bacterial populations are very small and a largesample is needed to get adequate numbers. The filter is then placed onthe surface of a chosen medium, incubated and the bacterial colonies arecounted and evaluated. This method is widely used and gives good resultsin general if combined with proper reagents and media, but is expensiveand time-consuming. The MF method can be used well in combination withthe new method described herein.

The Presence/Absence (P/A) test

This test, which involves the reagentsO-nitrophenyl-β-D-galactopyranoside (ONPG), a β-galactosidase substrateand 4-methylumbelliferyl-β-D-glucuronide (MUG), a β-glucuronidasesubstrate, results in the determination of the presence or absence ofgeneral coliforms and E. coli. The test relies on the fact thatgenerally all coliforms produce β-galactosidase, but only E. colistrains produce β-glucuronidase. If any coliforms are present, the brothmedium turns a yellow color due to the activity of galactosidase enzymeon the ONPG material causing the release of a diffusible yellow pigment.If E. coli is present, the broth medium will demonstrate a bluefluorescence when irradiated with ultraviolet rays due to the breakdownof the MUG reagent with the release of the fluorogenic dye caused by theproduction of the glucuronidase enzyme. These reactions are veryspecific and allow both general coliforms and E. coli to be identifiedin a single test in a single sample. But, since both reagents producediffusible pigments, the test has the disadvantage of not being directlyquantitative for either bacterial type.

The reagent 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) isa known test compound for identifying coliforms. When acted on by theβ-galactosidase enzyme produced by coliforms, X-gal forms an insolubleindigo blue precipitate. X-gal can be incorporated into a nutrientmedium such as an agar plate, and if a sample containing coliforms ispresent, the coliforms will grow as indigo blue colonies. X-gal has theadvantage over the compound ONPG, described above, in that it forms aninsoluble precipitate, rather than a diffusible compound, therebyallowing the quantitative determination of coliforms.

Recently, a similar compound, 5-bromo-4-chloro-3-indolyl-β-D-glucuronide(X-gluc) has been developed for the identification of E. coli. Whenacted on by the β-glucuronidase enzyme produced by E. coli, X-gluc formsan insoluble indigo blue precipitate. X-gluc has the advantages over thecompound MUG, described above, in that it forms an insolubleprecipitate, rather than a diffusible compound, thereby allowing thequantitative determination of E. coli. Further, it does not require theuse of ultraviolet light. X-gluc and its use to identify E. coli aredescribed in Watkins, et al, Appl. Environ. Microbiol. 54:1874-1875(1988). A similar compound, indoxyl-β-D-glucuronide, which also producessharp blue colonies of E. coli, was described in Ley, et al, Can. J.Microbiol. 34:690-693 (1987).

X-gal and X-gluc have the disadvantage that they each contain the exactsame chromogen and therefore they cannot be used together to identifyand distinguish between both E. coli and general coliforms in a singletest with a single sample. Both X-gal and X-gluc cause the formation ofidentically hued indigo blue colonies. A person using both reagentstogether would be able to quantitatively identify the total number ofcoliforms, the same as if X-gal were used alone, but would not be ableto tell which of the colonies were E. coli and which were othercoliforms besides E. coli.

SUMMARY OF THE INVENTION

A method has now been found for quantitatively identifying anddifferentiating microorganisms having β-galactosidase but notβ-glucuronidase activity and microorganisms having β-glucuronidaseactivity, comprising the steps of combining a chromogenicβ-galactosidase substrate capable of forming an insoluble precipitate ofa first color upon reacting with β-galactosidase, a chromogenicβ-glucuronidase substrate capable of forming an insoluble precipitate ofa second color contrasting with the first color upon reacting withβ-glucuronidase, and a nutrient base medium to form a test medium,inoculating the test medium with a sample to be tested for the presenceof microorganisms, incubating the test medium, examining the test mediumfor the presence of colonies of the first color, such colonies beingcolonies of microorganisms having β-galactosidase but notβ-glucuronidase activity, and the presence of colonies of the secondcolor, such colonies being colonies of microorganisms havingβ-glucuronidase activity, and enumerating the microorganisms havingβ-galactosidase but not β-glucurosidase activity and the microorganismshaving β-glucuronidase activity.

The advantages of the new method include the following. First, it allowsthe chromogenic differentiation between general coliforms, which haveβ-galactosidase activity, and E. coli, which additionally haveβ-glucuronidase activity, in the same test plate with the same sample.It is also quantitative so that exact counts of the numbers of viableorganisms of each type are determined. This is much more meaningful thanjust a presence/absence test as levels of contamination can bedetermined. The new method does not require any special apparatus orequipment such as a UV light source or special filter apparatus. The newmethod is based on enzymatic reactions rather than fermentationreactions which are more difficult to interpret and less precise.Because the new method does not require inhibitors, there is theadditional capability of quantifying the general microbial populationalong with general coliforms and E. coli. This is an important addedfeature.

A further aspect of this invention is the novel compound6-chloroindolyl-β-D-glucuronide, which forms an insoluble magentaprecipitate when reacted upon by β-glucuronidase that contrasts in colorwith the indigo blue precipitate formed by5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) by the actionof β-galactosidase. 6-Chloroindolyl-β-D-glucuronide may be used togetherwith X-gal in the method of this invention. General coliforms grow in amedium containing 6-chloroindolyl-β-D-glucuronide and X-gal as colonieshaving an indigo blue color whereas E. coli grow as colonies having apurplish or magenta color. Composition and test media having6-chloroindolyl-β-D-glucuronide and5-bromo-4-chloro-3-indolyl-β-D-galactoside for quantitativelyidentifying and differentiating general coliforms and E. coli are yetanother aspect of this invention.

A further aspect of this invention is the novel compound6-chloroindolyl-β-D-galactoside, which forms an insoluble magentaprecipitate when reacted upon by β-galactosidase that contrasts with theindigo blue precipitate formed by5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) orindoxyl-β-D-glucuronide by the action of β-glucuronidase.6-Chloroindolyl-β-D-galactoside may be used together with X-gluc orindoxyl-β-D-glucuronide in the method of this invention. Generalcoliforms grow in a medium containing 6-chloroindolyl-β-D-galactosideand either X-gluc or indoxyl-β-D-glucuronide as colonies having amagenta color whereas E. coli grow as colonies having an indigo bluecolor. Compositions and test media having6-chloroindolyl-β-D-galactoside and either5-bromo-4-chloro3-indolyl-β-D-glucuronide or indoxyl-β-D-glucuronide forquantitatively identifying and differentiating general coliforms and E.coli are yet another aspect of this invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a color photograph showing a test plate having a test mediumin accordance with the present invention.

The test medium includes 6-chloroindolyl-β-D-galactoside and5-bromo-4-chloro-3-indolyl-β-D-glucuronide. The test medium wasinoculated with a mixture of E. coli and general coliforms and wasincubated for 48 hrs. at 35° C. The photograph shows magenta-coloredcolonies, which are general coliforms and indigo blue-colored colonies,which are E. coli.

DETAILED DESCRIPTION OF THE INVENTION

The method of the present invention allows the quantitativeidentification and differentiation of microorganisms havingβ-galactosidase but not β-glucuronidase activity and microorganismshaving β-glucuronidase activity.

Microorganisms having β-galactosidase activity include those commonlyknown by the designation "coliform". There are various definitions of"coliform", but the generally accepted ones include bacteria which aremembers of the Enterobacteriaceae and have the ability to fermentlactose with gas production. The genera Citrobacter, Enterobacter,Klebsiella and Escherichia are the generally listed members of thecoliform group.

Microorganisms having β-glucuronidase activity primarily include onlythose strains of coliform of the species Escherichia coli.

As used in this application, the term "general coliforms" refers tocoliforms other than E. coli. These are microorganisms havingβ-galactosidase activity and not having β-glucuronidase activity.

The chromogenic β-galactosidase substrate is a β-galactoside comprisinggalactose joined by β-linkage to a substituent that forms an insolubleprecipitate of a first color when liberated by the action ofβ-galactosidase on the substrate.

The chromogenic β-glucuronidase substrate is a β-glucuronide comprisingglucuronic acid joined by β-linkage to a substituent that forms aninsoluble precipitate of a second color, contrasting with the firstcolor, when liberated by the action of β-glucuronidase on the substrate.The β-glucuronidase substrates and compounds described herein as"glucuronides" include carboxylate salts formed by reacting a suitablebase with the glucuronic carboxyl group. Suitable bases include alkalimetal or alkaline earth metal hydroxides or carbonates, for example,sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesiumhydroxide, and corresponding carbonates; and nitrogen bases such asammonia and alkylamines such as trimethylamine, triethylamine andcyclohexylamine.

The β-galactosidase substrate and the β-glucuronidase substrate areselected so that the precipitates formed by each are of contrastingcolors so that colonies of microorganisms having β-galactosidase but notβ-glucuronidase activity and colonies of microorganisms havingβ-glucuronidase activity can be visually distinguished. The exact colorof each type of microorganism colony is not crucial as long as each typecan be distinguished. The precipitates should be insoluble in the testmedium so that the colonies of microorganisms producing each precipitatecan be visually counted. Further, it will be readily appreciated thatthe β-galactosidase substrate and β-glucuronidase substrates should becompounds that are approximately colorless or are not deeply colored, sothat they do not interfere with the detection of the colored insolubleprecipitates produced by the action of β-galactosidase andβ-glucuronidase. The substrates should be compounds that can be madesoluble in the test medium. The determination of whether a givenβ-galactoside or β-glucuronide is operable can be made by a simple testcomprising incubating the β-galactoside or β-glucuronide in an agar orpectin test medium inoculated with general coliforms or E. coli andobserving whether colored colonies grow in the test medium. Thedetermination of whether a given β-galactoside and β-glucuronide can beused together in the method of this invention and fall within the scopeof the invention can be made by testing the two compounds together in atest medium inoculated with a mixture of both general coliforms and E.coli and observing whether the colonies of E. coli and the colonies ofgeneral coliforms can be visually differentiated by a contrast in colorof each type of colony.

As an example of suitable compounds for the practice of the method ofthis invention, the compound5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) is acommercially available β-galactosidase substrate that produces aninsoluble precipitate having an approximately indigo blue color whenreacted upon by β-galactosidase. Possible β-glucuronidase substratesthat could be used with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranosidewould be ones that produce an insoluble precipitate having a color suchas red or yellow that contrasts with indigo blue and is not totallymasked by the blue color. An example is the novel compound6-chloroindolyl-β-D-glucuronide. This produces an insoluble precipitatehaving a magenta color contrasting with and visually distinguishablefrom indigo blue. The preparation of this compound is described below.

The compound 5-bromo-4-chloro-3-indolyl-β-D-glucuronide is acommercially available β-glucuronide substrate that produces aninsoluble precipitate having an approximately indigo blue color whenreacted upon by β-glucuronidase. Indoxyl-β-D-glucuronide is a similarcompound, the preparation of which is described in Ley et al., Lan J.Microbiol. 39:690-693 (1987), the disclosure of which is herebyincorporated by reference. Possible β-galactosidase substrates thatcould be used with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide orindoxyl-β-D-glucuronide would be ones that produce an insolubleprecipitate having a color such as red or yellow that contrasts withindigo blue. An example is the novel compound6-chloroindolyl-β-D-galactoside. This produces an insoluble precipitatehaving a magenta color contrasting with and visually distinguishablefrom indigo blue. The preparation of this compound is described below.

Preparation of Novel β-galactosidase and β-glucuronidase Substrates

The following illustrates the synthesis of6-chloroindolyl-β-D-galactoside:

1. 4-Chloroanthranilic acid

4-Chloroanthranilic acid was prepared from 5-chloro-2-methylaniline asdescribed by James R. Piper and Frank J. Stevens in "SubstitutedIndole-3-acetic acids by the Reformatsky Reaction" and published in theJournal of Organic Chemistry, Volume 27, pp. 3134-3137.

A solution of 70.8 g (0.5 M) 5-chloro-2-methylaniline in 70 ml glacialacetic acid was treated with 52 g (0.51 M) acetic anhydride and broughtto gentle reflux for 30 minutes. The solution was poured into 400 mlcold water and the n-acetyl-5-chloro-2-methylaniline that separated wasfiltered off and washed with cold water.

The damp acetyl derivative was suspended in 2 L 0.25 M magnesium sulfatesolution at 85° C. With vigorous stirring, 240 g potassium permanganateas added, portionwise, over a 1.5 hour period, keeping the temperaturebetween 85° and 90° C. The mixture was filtered hot, and the manganesedioxide by-product was washed with 1 L hot water. The aqueous filtratewas cooled to room temperature and acidified with 20% sulfuric acid to apH of 1, precipitating n-acetyl-4-chloroanthranilic acid. The productwas filtered off and washed with cold water.

The damp n-acetyl-4-chloroanthranilic acid was suspended in 400 ml ofconcentrated hydrochloric acid (12 M) and stirred and heated at 80° C.for 8 hours. The mixture was cooled to 10° C. and the resulting4-chloroanthranilic acid hydrochloride was filtered off. The solid wassuspended in 300 ml water and sodium acetate was added, portionwiseuntil the pH was 5. The resulting 4-chloroanthranilic acid was filteredoff and recrystallized from the minimum of hot ethanol. The final yieldof off-white crystals was 50 g, melting at 239°-240° C.

2. 5-Chloro-2-carboxyphenylglycine sodium salt

4-Chloroanthranilic acid weighing 474 g (2.69 M) was suspended in 1200ml water in a 5 L flask. A 30% potassium hydroxide solution was added,slowly, with stirring, until the pH was 7.0 to 8.0. The4-chloro-anthranilic acid dissolved to form a solution of the potassiumsalt. To this solution, was added a solution of 326.5 g (2.80 M) ofsodium monochloroacetate in 800 ml water. The resultant solution wasthen placed in a pressure bottle and allowed to stand at 60° C. forthree days. The product precipitated and the mixture was almost solidafter three days. The product was filtered off and washed with 200 mlice cold water. After drying in vacuo, the off white product weighed 335g (43% yield) and melted at 278°-280° C.

3. 6-Chloroindoxy-1,3-diacetate

Into a 5 L, 3-neck flask equipped with mechanical stirring, refluxcondenser and gas evolution bubbler was placed 335 g (1.33 M) of5-chloro-2-carboxyphenylglycine sodium salt, 2.3 1 acetic anhydride and421 g of anhydrous sodium acetate. The mixture was brought to reflux andmaintained for 3 hours until the evolution of carbon dioxide was nearlycomplete. The mixture was placed in a beaker while hot and chilled to 0°C. overnight. The next day, the product was filtered off and mixed with2 L water and stirred for 1 hour to hydrolyze any residual aceticanhydride. The solid was filtered off and washed with water and thendried in vacuo. This crude material, when dry, was dissolved in theminimum of hot ethyl acetate and allowed to crystallize overnight at 0°C. After filtering and washing with a little cold hexane, and drying invacuo, the product weighed 150 g and had a melting point of 112°-113° C.

4. 6-Chloro-N-acetylindol-3-ol

A solution of 572 ml of concentrated sulfuric acid was added to 63 ml ofwater with stirring and cooling. When the acid solution was at roomtemperature, 134 g (0.53 M) of 6-chloroindoxyl-1,3-diacetate was added,portionwise, with stirring over a period of one hour keeping thetemperature of the solution between 20° to 25° C. After stirring for 30additional minutes after the addition was complete, the solution waspoured onto 2 kg ice. The ice was allowed to melt and the insolubleproduct was filtered off and washed with cold water. This product wasthen protected from light and air and dried in a vacuum oven at roomtemperature. The yield of light yellow solid was 110 gm. This productwas very unstable and could not be stored for long period withoutdecomposition. It was used immediately in the next step.

5.6-Chloroindolyl-β-D-galactoside pentaacetate

Into a 1 L, 3-neck flask, equipped with a mechanical stirrer and a gassparger tube was added 250 ml dry methanol, followed by 2.3 g (0.1 M) ofsodium metal. While the sodium was reacting, a nitrogen sparge wasstarted at the rate of 1 bubble per second. When the sodium haddissolved, the solution was cooled to 0° C. and 20.9 g (0.1 M) of6-chloro-N-acetylindol-3-ol was added, maintaining nitrogen purgethroughout. After stirring for 10 minutes, 41.1 g (0.1 M) ofacetobromogalactose was added all at once. The reaction was allowed toproceed for 5 hours, slowly warming up to room temperature. Theby-product of sodium bromide was filtered off and the solution wasevaporated in vacuo to leave a gum. This gum was dissolved in ethylacetate and then extracted with water in a separatory funnel. Theorganic phase was dried over sodium sulfate, filtered and evaporated invacuo once again. The residue was dissolved in the minimum of hotethanol and allowed to cool slowly to room temperature. The productcrystallized as fine white needles and was filtered off and dried. Theyield was 18 g melting at 165°-166° C.

6. 6-Chloroindolyl-β-D-galactoside

6-Chloroindolyl-β-D-galactoside pentaacetate 10 g (0.018 M) wassuspended in 250 ml of dry methanol. To the suspension was added asolution of 100 mg sodium metal dissolved in 10 ml dry methanol. Themixture immediately formed a clear solution. After standing for 1 hourat room temperature, the solution was evaporated in vacuo to a lowvolume and stored at 0° C. overnight. The next day, the crystallineproduct was filtered off and washed with ethyl ether and dried in vacuo.The yield was 2.5 g white crystals that melted at 100°-102° C.

The following illustrates the synthesis of6-chloroindolyl-β-D-glucuronic acid monocyclohexylammonium salt.

Steps 1-4 are identical to the above steps 1-4 in the synthesis of6-chloroindolyl-β-D-galactoside.

5.Methyl[6-chloro-N-acetylindol-3-yl-(2,3,4-tri-O-acetyl-β-D-glucopyranoside)]uronate:

To 100 ml of dry methanol at -5° C. containing 1.8 g of sodium metal wasadded 16.34 g (0.078M) of 6-chloro-N-acetylindol-3-ol under anatmosphere of nitrogen. The solution was cooled and maintained at 0° C.while adding a solution of 31 g (0.08 M) of1-bromo-1-deoxy-2,3,4-tri-O-acetyl-α-D-glucopyranuronate in 150 ml ofdry methanol all at once. The mixture was allowed to warm to roomtemperature while stirring under nitrogen for four hours. The mixturewas then sparged with air and unreacted indoxyl was converted toinsoluble indigo. The mixture was filtered and the filtrate wasevaporated to a gum in vacuo. The gummy residue was dissolved in ethylacetate (approx. 200 ml) and washed in a separatory funnel with 200 mlwater. The organic phase was separated, filtered and evaporated to agum. The residue was taken up in 28 ml of acetic anhydride and 40 ml ofpyridine and allowed to stand at room temperature overnight. Thesolution was poured into 600 ml water and allowed to stand until theproduct had solidified. The product was filtered off and crystallizedfrom the minimum of boiling ethanol. White crystals, melting at 172° C.were obtained. The yield was 11 grams.

6. 6-Chloroindolyl-β-D-glucopyranoside uronate monocyclohexylammoniumsalt

To 400 ml of acetone containing 10 g ofmethyl-(6-chloroindol-3-yl-2,3,4-tri-O-acetyl-β-D-glucopyranoside)uronate was added 100 ml of 1 N sodium hydroxide. The solution wasstirred at room temperature for 20 minutes and then evaporated in vacuoto approximately 150 ml volume. The solution was adjusted to pH 7.5 withdilute acetic acid and then treated with stirring with a saturatedsolution of lead acetate. The lead salt of the product was collected byfiltration and immediately suspended in 100 ml of methanol. To thisstirred suspension of lead salt was added hydrogen sulfide gas until thelead was converted into lead sulfide. The lead salt was removed byfiltration and the filtrate containing the product was evaporated toapproximately 50 ml in vacuo. A solution of 1 ml of cyclohexylamine in10 ml methanol was added and upon cooling the amine salt started tocrystallize. The methanol suspension of product was diluted with onevolume of ethyl ether and filtered. The product consisted of 2 g ofoff-white crystals melting at 211°-213° C.

Other novel β-galactosidase and β-glucuronidase substrates

Other β-galactosides and β-glucuronides that may be useful in the methodof this invention include those that fall into the general category ofsubstituted indolyl β-galactosides and β-glucuronides. While it is notintended to limit this invention to any particular theory or mechanism,it is believed that when β-galactosidase or β-glucuronidase substrateshaving substituted indolyl substituents are reacted upon by theirrespective enzymes, the substituted indolyl substituents released by theaction of the enzyme convert in situ to insoluble indigo analogues. Forexample, when 6-chloroindolyl-β-D-galactoside is reacted upon byβ-galactosidase, the released 6-chloroindolyl reacts with itself andforms 6,6'-dichloroindigo, a magenta insoluble precipitate. Thissuggests that other compounds similar to 6-chloroindolyl-β-D-galactosideor 6-chloroindolyl-β-D-glucuronide could be made based upon symmetricalindigo analogues having a color similar to 6,6'-dichloroindigo. Thesynthesis and absorption spectra of symmetrical chloroindigos werereported in Sadler et al., JACS 78-1251-1255 (1956), the disclosure ofwhich is incorporated herein by reference. It appears therein that thecompounds 4,4',6,6' tetrachloroindigo, 6,6' ,7,7' tetrachloroindigo, and4,4',6,6',7,7' hexachloroindigo are similar in color to6,6'-dichloroindigo. Thus, the respective β-galactosides namely4,6-dichloroindolyl-β-D-galactoside,6,7-dichloroindolyl-β-D-galactoside, and4,6,7-trichloroindolyl-β-D-galactoside could be made and used asβ-galactosidase substrates in the same manner as 6-chloroindolylβ-D-galactoside. Similarly, the respective β-glucuronides, namely4,6-dichloroindolyl-β-D-glucuronide,6,7-dichloroindolyl-β-D-glucuronide,4,6,7-trichloroindolyl-β-D-glucuronide, could be made and used asβ-glucuronidase substrates in the same manner as6-chloroindolyl-β-D-glucuronide.

Preparation of Test Medium

The test medium is formed by combining the β-galactosidase substrate andthe β-glucuronidase substrate with a nutrient base medium. The nutrientbase medium can be any culture medium known in the art for growingmicroorganisms. Generally such media include growth nutrients, buffers,water, and a gelling agent. Possible gelling agents include agars,pectins, carrageenans, alginates, locust bean, and xanthins, amongothers.

The following is an example of the preparation of a test medium suitablefor use in this invention.

To prepare sufficient quantity of the β-galactosidase substrate and theβ-glucuronidase substrate for one liter of medium, 150 mg of theβ-galactosidase substrate and 75 mg of the β-glucuronidase substrate areweighed and added to 5 ml of dimethylformamide (DMF). The mixture isagitated until dissolved. An additional 10 ml of deionized water isadded mixed. The mixture is filter sterilized with a micropore filter.

Standard agar medium may be made by adding 15 gm of bacteriologicalquality agar gum to the following nutrient formula

    ______________________________________                                        Pancreatic Digest of Casein                                                                           5.0    gm                                             Yeast Extract           3.0    gm                                             Dipotassium Phosphate   0.3    gm                                             Deionized Water         1000   ml                                             ______________________________________                                         and then sterilizing at 121° C. for 15 minutes. The medium should     be adjusted to result in a pH of 7.0. The sterilized agar medium is     allowed to drop to a temperature of 45° C. in a water bath and then     the sterile solution containing 150 mg of the β-galactosidase     substrate and 75 mg of the β-glucuronidase substrate is added. The     medium is mixed thoroughly and poured into sterile petri plates at a     volume of 20 ml/plate.

A pectin-based test medium may be prepared using the same stepsdescribed above except that 25 gm of low methoxyl pectin is used as thesolidifying agent and the medium is poured at room temperature intopetri plates containing a thin gel layer containing calcium ions whichcombine with the pectin to form a solid gel. A suitable pectin culturemedium is described in U.S. Pat. No. 4,241,186 and U.S. Pat. No.4,282,317, the disclosures of which are incorporated herein byreference. A pectin-based medium is preferred over a standard agarmedium because it has the advantages of convenience and temperatureindependence for the user. The use of pectin media is well described andaccepted as a result of AOAC collaborative studies and other publishedand in-house investigations.

A suitable pectin medium is commercially available from RCR Scientific,Inc. under the trademark Redigel.

Inoculation of the Test Medium with the Sample

The test medium may be inoculated with a sample to be tested for thepresence of microorganisms having β-galactosidase but not glucuronidaseactivity and microorganisms having β-glucuronidase activity by anymethod known in the art for inoculating a medium with a samplecontaining microorganisms. For example, the sample to be tested may beadded to the petri plates prior to adding the nutrient base medium (pourplate technique) or spread on the surface of the plates after they havecooled and solidified (swab or streak plate technique).

Incubation of the Test Medium

The inoculated test medium is incubated for a sufficient time and atsuch a temperature for individual microorganisms present in the sampleto grow into detectable colonies. Suitable incubation conditions forgrowing microorganisms in a medium are known in the art. Preferably, thetest medium is incubated for about 24-48 hours at a temperature of about30°-40° C.

Unless inhibitors of the general microbial population are used, thegeneral microbial population as well as general coliforms and E. coliwill grow in the incubated test medium. Because microorganisms otherthan general coliforms and E. coli rarely produce β-galactosidase orβ-glucuronidase, the general microbial population will show as it doeson a standard agar pour plate as white or colorless colonies. Generalcoliforms produce β-galactosidase, which acts upon the β-galactosidasesubstrate in the test medium, causing the β-galactosidase substrate toform an insoluble precipitate having a color in accordance with theparticular β-galactosidase substrate used. Because the precipitateformed is insoluble in the test medium, it remains in the immediatevicinity of microorganisms producing the β-galactosidase. As themicroorganisms reproduce to form colonies, the colonies show as colonieshaving the color produced by the β-galactosidase substrate.

E. coli produces β-galactosidase, but, unlike general (other) coliforms,also produces β-glucuronidase. Insoluble precipitates of both theβ-galactosidase substrate and β-glucuronide substrate are formed by theaction of the respective enzymes. The colonies of E. coli show ascolonies having a color different from and contrasting with the color ofthe colonies of general coliforms because of the presence of thecontrastingly colored insoluble precipitate of the β-glucuronidasesubstrate. Preferably, the β-galactosidase substrate and theβ-glucuronidase substrate are selected so that the β-glucuronidasesubstrate produces an insoluble precipitate that is darker in color thanthe insoluble precipitate produced by the β-galactosidase substrate.This would allow the precipitate produced by the β-glucuronidasesubstrate to mask the precipitate produced by the β-galactosidasesubstrate in colonies of E. coli and would make it easier for coloniesof E. coli to be differentiated from colonies of general coliforms.Preferably 6-chloroindolyl-β-D-galactoside is used with5-bromo-4-chloro-3-indolyl-β-D-glucuronide or indoxyl-β-D-glucuronide.

Examination of the Test Medium and Enumeration of Microorganisms

Since the β-galactosidase substrate and the β-glucuronidase substrateare selected so that the colors of the precipitate produced by eachcontrast with each other, the colonies of each type of coliform, generalcoliforms and E. coli, can be readily differentiated by visual means.For example, if 6-chloroindolyl-β-D-galactoside is used as theβ-galactosidase substrate and 5-bromo-4-chloro-3-indolyl-β-D-glucuronideis used as the β-glucuronide substrate, general coliforms will show asmagenta-colored colonies and E. coli as indigo blue-colored colonies.

The colonies of each type of microorganism may be enumerated by countingthe colonies or by other methods known in the art for enumeratingmicroorganisms on a test plate. The number of colonies of each typeindicates the number of microorganisms of each type originally presentin the sample before incubation.

Optional Ingredients Inhibitors

The method of the present invention does not require inhibitors.However, the medium may be made more selective for general coliforms andE. coli if desired by the addition of various compounds that areinhibitory to the general microbial population, but have little or noeffect on coliforms. Following are some compounds which may be used: a)bile salts, about 0.3 g/liter, b) sodium lauryl sulfate, about 0.2g/liter, c) sodium desoxycholate, about 0.2 g/liter, d) Tergitol 7,about 0.1 ml/liter. The use of one or more of these compounds reducesthe background (non-coliform) microorganism presence and makes a lesscluttered plate and eliminates the possibility of inhibition orinterference by the non-coliform organisms in the sample.

Inducers

It is possible that the enzyme production of the general coliforms maybe enhanced by the addition to the medium formulations of very smallamounts of substances known as enzyme inducers. The specific inducer forβ-galactosidase is available and is known chemically asisopropyl-β-D-thiogalactopyranoside. Adding approximately 25 mg/liter ofmedium has a positive and noticeable effect on the speed of enzymeproduction for some species of coliforms.

EXAMPLE 1

6-Chloroindolyl-8-D-galactoside and5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) were each testedseparately with 17 different bacterial species including β-galactosidaseproducing microorganisms, β-glucuronidase producing microorganisms, andmicroorganisms which produce neither enzyme.

As shown in Table I below, only E. coli grew as indigo blue colonies inthe X-gluc medium (as indicated by a +in the X-gluc column). E. coli,Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii andKlebsiella pneumoniae grew as magenta colonies in the medium containing6-chloroindolyl-β-D-galactoside (as indicated by a +in this column). Allthe other species grew as colorless colonies on both media, as would beexpected (as indicated by a -in the respective columns).

                  TABLE I                                                         ______________________________________                                                          6-chloroindolyl-                                            Species           β-D-galactoside                                                                      X-gluc                                          ______________________________________                                        Acinetobacter calcoaceticus                                                                     -           -                                               Bacillus cereus   -           -                                               Bacillus megaterium                                                                             -           -                                               Bacillus subtilus -           -                                               Citrobacter freundii                                                                            +           -                                               Enterobacter aerogenes                                                                          +           -                                               Enterobacter cloacae                                                                            +           -                                               Escherichia coli  +           +                                               Klebsiella pneumoniae                                                                           +           -                                               Proteus vulgaris  -           -                                               Pseudomonas aeruginosa                                                                          -           -                                               Salmonella typhimurium                                                                          -           -                                               Sarcina lutea     -           -                                               Serratia marcescens                                                                             -           -                                               Staphylococcus aureus                                                                           -           -                                               Staphylococcus epidermidis                                                                      -           -                                               Staphylococcus faecalis                                                                         -           -                                               ______________________________________                                    

EXAMPLE 2

Test plates containing both 6-chloroindolyl-β-D-galactosidase and5-bromo-4-chloro-3-indolyl-β-D-glucuronidase in a pectin gel medium wereprepared. The test plates were inoculated with natural river water orwith mixes of E. coli and other coliforms (E. aerogenes and K.pneumoniae).

Agar and pectin (Redigel) test plates were also prepared for the VioletRed Bile (VRB) test for coliforms using standard procedures. Theseplates were also inoculated with river water or mixes of E. coli andother coliforms.

The test plates were incubated and then examined. The results are shownin Table II below. The numbers in each column represent the number ofcolonies of each type per test plate. The number of colonies of eachtype per test plate ultimately corresponds to the number of individualmicroorganisms of each type present in the sample before incubation.

                  TABLE II                                                        ______________________________________                                        Mixture of 6-chloro-                                                          indolyl-β-D-galactoside                                                  and 5-bromo-4-chloro-3-                                                       indolyl-β-D-glucuronide                                                                     VRB Agar   VRB Redigel                                     Red        Blue    Total   Total Count                                                                            Total Count                               ______________________________________                                        RUN 1                                                                         ______________________________________                                        River 1                                                                              260     --      260   290      325                                     River 2                                                                              305      5      310   290      350                                     Mix 1  28      10      38     5       41                                      Mix 2  31       6      37     7       24                                      ______________________________________                                        Mix 1 and 2 are E. coli (blue) and Enterobacter                               aerogenes (red)                                                               ______________________________________                                        RUN 2                                                                         ______________________________________                                        River 1                                                                               4      31      36             38                                      River 2                                                                               4      42      46             26                                      Mix 1  10      70      80             90                                      Mix 2   7      90      97             90                                      ______________________________________                                        Mix 1 and 2 are E. coli (blue) and Klebsiella                                 pneumoniae (red)                                                              ______________________________________                                    

As shown above, the total counts of coliforms indicated by the testmedium of the present invention are in approximate agreement with theresults obtained with the VRB tests. However, the present method has theadvantage of differentiating E. coli from general coliforms, so that aseparate total of each may be obtained. With VRB, further tests would berequired to determine if any of the colonies were E. coli, requiringfurther time and expense.

EXAMPLE 3

A test plate containing 6-chloroindolyl-β-D-galactosidase and5-bromo-4-chloro-3-indolyl-β-D-glucuronide in a pectin gel medium wasinoculated with a sample containing E. coli and general coliforms. Theplate was incubated for 48 hours at 35° C. and then examined for thepresence of magenta-colored colonies and indigo blue-colored colonies.FIG. 1 is a photograph of the test plate as it appeared after 48 hours.As shown in FIG. 1, the indigo blue colonies of E. coli are easilydistinguished by visual means from magenta colonies of generalcoliforms. The number of colonies of each type of microorganisms iseasily counted--there are 13 colonies of E. coli and 52 colonies ofgeneral coliforms.

Although the invention has been described in considerable detail withspecific reference to certain advantageous embodiments thereof,variations and modifications can be made without departing from thescope of the invention as described in the specification and defined inthe appended claims.

What is claimed is:
 1. A method for testing the presence of and quantitatively identifying and differentiating general coliforms and E. coli in a test sample, comprising the steps ofinoculating a test medium substrate capable of forming a solid matrix or solid surface with said test sample, said test medium comprising a chromogenic β-galactosidase substrate capable of forming a first water insoluble precipitate of a first color upon reacting with β-galactosidase, a chromogenic β-glucuronidase substrate capable of forming a second water insoluble precipitate of a second color contrasting with said first color upon reacting with β-glucuronidase, and a nutrient base medium, said first and second colors being visibly distinguishable in daylight, incubating said test medium as a solid to produce colonies of said general coliforms and E. coli and first and second colored precipitates corresponding to said colonies, examining said test medium for the presence of colonies having said first color, such colonies being colonies of general coliforms having β-galactosidase but not β-glucuronidase activity, and the presence of colonies having said second color, such colonies being colonies of E. coli having β-glucuronidase activity, and enumerating said colonies having β-galactosidase activity and said colonies having β-glucuronidase activity.
 2. The method according to claim 1, whereinsaid chromogenic β-galactosidase substrate is 6-chloroindolyl-β-D-galactoside, and said chromogenic β-glucuronidase substrate is 5-bromo-4-chloro-3-indolyl-β-D-glucuronide.
 3. The method according to claim 1, whereinsaid chromogenic β-galactosidase substrate is 6-chloroindolyl-β-D-galactoside, and said chromogenic β-glucuronidase substrate is indoxyl-β-D-glucuronide.
 4. The method according to claim 1, whereinsaid chromogenic β-galactosidase substrate is 5-bromo-4-chloro-3-indolyl-β-D-galactoside, and said chromogenic β-glucuronidase substrate is 6-chloroindolyl-β-D-glucuronide.
 5. The method of claim 2, further comprising the steps ofcombining said 5-bromo-4-chloro-3-indolyl-β-D-glucuronide and a chromogenic indolyl β-galactosidase substrate capable of forming a water insoluble precipitate of a color contrasting with indigo blue upon reacting with β-galactosidase with said nutrient base to form a solid test medium.
 6. The method of claim 1, wherein said β-galactosidase is5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, said method comprising combining said β-galactosidase and a chromogenic indolyl β-glucuronidase substrate capable of forming a water insoluble precipitate of a color contrasting with indigo blue upon reacting with β-glucuronide with said nutrient base and forming said solid test medium.
 7. The method according to claim 6, whereinsaid chromogenic β-glucuronide substrate is 6-chloroindolyl-β-D-glucuronide.
 8. A method for testing the presence of and quantitatively identifying and differentiating general coliforms and E. coli in a test sample, said method comprising the steps of inoculating a test medium capable of forming a solid matrix or solid surface with said test sample to be tested for the presence of general coliforms or E. coli said test medium comprising a chromogenic β-galactosidase substrate capable of forming a first water insoluble precipitate of a first color upon reacting with β-galactosidase, a chromogenic indolyl β-glucuronidase substrate capable of forming an insoluble precipitate of a second color contrasting with said first color upon reacting with β-glucuronidase, and a nutrient base medium, wherein said indolyl β-glucuronidase is at least one selected from the group consisting of 6-chloroindolyl-β-D-glucuronide, 4,6-dichloroindolyl-β-D-glucuronide, 6,7-dichloroindolyl-β-D-glucuronide, 4,6,7-trichloroindolyl-β-D-glucuronide and salts thereof, andincubating said test medium as a solid to produce colonies of said general coliforms and E. coli and said first and second colored precipitates corresponding to said colonies, and enumerating said colonies.
 9. A method according to claim 8, wherein after incubating said test medium, said method further comprisesexamining said test medium for the presence of colonies having said first color, such colonies being colonies of microorganisms having β-galactosidase but not β-glucuronidase activity, and the presence of colonies having said second color, such colonies being colonies of microorganisms having β-glucuronidase activity, and enumerating said colonies of microorganisms having β-galactosidase activity and said colonies of microorganisms having β-glucuronidase activity.
 10. A method according to claim 8, wherein said test medium is capable of forming a solid, said method comprising inoculating said test medium with said test sample and solidifying said test medium to a solid before incubating.
 11. A method for testing the presence of and quantitatively identifying and differentiating general coliforms and E. coli in a test sample, said method comprising the steps ofinoculating a solid or gel test medium with a sample to be tested for the presence of general coliforms or E. coli, said solid test medium comprising a chromogenic indolyl β-galactosidase substrate capable of forming an insoluble precipitate of a first color upon reacting with β-galactosidase, a chromogenic β-glucuronidase substrate capable of forming an insoluble precipitate of a second color contrasting with said first color upon reacting with β-glucuronidase, and a nutrient base medium wherein said indolyl β-galactosidase is at least one selected from the group consisting of 6-chloroindolyl β-D-galactoside, 4,6-dichloroindolyl-β-D-galactoside, 6,7-dichloroindolyl-β-D-galactoside, 4,6,7-trichloroindolyl-β-D-galactoside and salts thereof, incubating said test medium to produce colonies of general coliforms and E. coli and said first and second colored precipitates corresponding to said colonies, and enumerating said colonies.
 12. A method according to claim 11, wherein after incubating said test medium, said method further comprisesexamining said test medium for the presence of colonies having said first color, such colonies being colonies of microorganisms having β-galactosidase but not β-glucuronidase activity, and the presence of colonies having said second color, such colonies being colonies of microorganisms having β-glucurosidase activity, and enumerating said colonies of microorganisms having β-galactosidase activity and said colonies of microorganisms having β-glucuronidase activity.
 13. A method according to claim 11, wherein said β-glucuronidase is at least one selected from the group consisting of 6-chloroindolyl-β-D-glucuronide, 4,5-dichloroindolyl-β-D-glucuronide. 6,7-dichloroindolyl-β-D-glucuronide, 4,6,7-trichloroindolyl-β-D-glucuronide and salts thereof.
 14. A method according to claim 11, wherein said test medium is capable of forming a solid and said method comprises inoculating said test medium with said test sample and solidifying said test medium before incubating.
 15. A method for detecting the presence of E. coli having β-glucuronidase activity in a test sample, said method comprising the steps ofinoculating a test medium with said test sample to be tested for the presence of microorganisms, said test medium being a solid or a liquid capable of forming a solid comprising a chromogenic indolyl β-glucuronidase substrate capable of forming an insoluble precipitate upon reacting with β-glucuronidase, and a nutrient base medium, wherein said indolyl β-glucuronidase is at least one selected from the group consisting of 6-chloroindolyl-β-D-glucuronide, 4,6-dichloroindolyl-β-D-glucuronide, 6,7-dichloroindolyl-β-D-glucuronide, 4,6,7-trichloroindolyl-β-D-glucuronide and salts thereof, solidifying said test medium when said test medium is a liquid, incubating said test medium to produce colonies of E. coli and enumerating said colonies.
 16. A method for detecting the presence of general coliforms having β-galactosidase but not β-glucuronidase activity, said method comprising the steps ofinoculating a test medium with a sample to be tested for the presence of microorganisms, said test medium being a solid or a liquid capable of forming a solid comprising a chromogenic indolyl β-galactosidase substrate capable of forming an insoluble precipitate of a first color upon reacting with β-galactosidase, and a nutrient base medium, wherein said indolyl β-galactosidase substrate is at least one selected from the group consisting of 6-chloroindolyl β-D-galactoside, 4,6-dichloroindolyl-β-D-galactoside, 6,7-dichloroindolyl-β-D-galactoside, 4,6,7-trichloroindolyl-β-D-galactoside and salts thereof, solidifying said test medium when said test medium is a liquid, and incubating said test medium to produce colonies of said coliforms and enumerating said colonies. 